Four-dimensional imaging of living chondrocytes in cartilage using
confocal microscopy : a pragmatic approach.
Errington, Rachel J., Mark D. Fricker, Julian L. Wood, Andrew C. Hall,
and Nick S. White.
Department of Physiology, University of Oxford, Parks Road, OX1
3PT, Department of Plant Sciences, University of Oxford, South Parks
Road, OX1 3RB
APStracts 3:0304C, 1996.
Regulation of cell volume is a fundamental cellular homeostatic
mechanism in the face of osmotic stress. In normal articular
cartilage, chondrocytes are exposed to a changing osmotic
environment. We present a comprehensive protocol for studying the
volume regulatory behaviour of chondrocytes within intact cartilage
tissue using confocal laser scanning microscopy. Our data acquisition
regime optimises both signal-to-noise and cell viability during
timelapsed 3-D (x,y,z,t) imaging. The porcine cartilage is treated as
an integrated component of the imaging system and we demonstrate
methods for the direct assessment of tissue-induced axial attenuation
and image distortion. Paramatised functions describing these two
components of image degradation are used to correct experimental
data. The current study also highlights the problems associated with
the analysis and visualisation of 4-D images. We have devised two new
types of data reconstruction. The first compresses each 3-D timepoint
into a single quantitative view, termed a coordinate view. From these
reconstructions we are able to simultaneously view and extract cell
measurements. A second type, a 4-D reconstruction, uses colour to
represent relative changes in cell volume, again while maintaining
the morphological and spatial information. Both these approaches of
image analysis and visualisation have been implemented to study the
morphology, spatial distribution and dynamic volume behaviour of
chondrocytes following osmotic perturbation. We have mapped
chondrocyte shape, arrangement and absolute volume in situ which vary
significantly from the tissue surface through to the underlying bone.
Despite the rigid nature of the extracellular matrix, cartilage cells
are osmotically sensitive and respond to stimulation of volume
regulatory mechanisms. The combined techniques of CLSM and vital cell
labelling have enabled us to study, for the first time, the response
of chondrocytes in situ to changes in interstitial osmotic pressure.
Received 3 June 1996; accepted in final form 13 September 1996.
APS Manuscript Number C310-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 5 November 1996